In order to evade host immunity, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes unique endoglycosidases, including EndoS, which removes complex-type glycans in a highly specific manner from human IgG antibodies, and its homolog EndoS2, which can additionally remove IgG-linked high-mannose glycans. This renders antibodies incapable of eliciting host effector functions through either complement or Fc ? receptors (Fc?Rs), providing the bacteria with a survival advantage. Because antibodies are central players in many human immune responses and bridge the innate and adaptive arms of immunity, the analysis and manipulation of the enzymatic activities of EndoS and EndoS2 impact diverse fields in biomedicine. In particular, modifying antibody glycan structures can have significant impacts on their abilities to bind to Fc?Rs and the subsequent immune system reactions that they induce. The next generation of therapeutic antibodies is already being constructed with modified glycan chemistries to tailor their immune reactions and to increase their clinical potency. EndoS and EndoS2, as antibody-specific glycosidases, and glycosynthases derived thereof, are key enzymes in the future of antibody engineering. We propose that if the molecular mechanisms by which diverse endoglycosidases specifically recognize and hydrolyze distinct glycoprotein substrates are better understood that EndoS and EndoS2 variants can be rationally engineered to create a new class of antibody-modifying enzymes endowed with unique glycan specificities in order to modify antibodies that exhibit enhanced clinical properties. In this proposal, we will address three Specific Aims: (1) to determine the molecular basis of glycan specificity by endoglycosidases; (2) to define the role of carbohydrate binding modules ? non-enzymatic protein domains with glycan binding properties ? in endoglycosidase specificity and activity; and (3) to elucidate the molecular basis of protein specificity by endoglycosidases. Progress towards these complementary, yet independent, Specific Aims will significantly advance our understanding of glycan-modifying enzymes. Leveraging this knowledge in the context of EndoS and EndoS2 will enhance our ability to customize antibodies, further unleashing their vast therapeutic utility and expanding their positive impact on human health.